One of the most frequent reasons adult women seek medical treatment is for abnormal vaginal discharge and related symptoms. In women who visit their physician with a vaginal complaint, approximately 40% are diagnosed as having some form of vaginitis, and 90% of these cases are either: bacterial vaginosis (BV), trichomoniasis or vulvovaginal candidiasis. The most common among these is bacterial vaginosis. BV is associated with placental infection, premature delivery and low birth weight babies, increased septicemia, premature membrane rupture and episiotomy infection. As such, the need for rapid, accurate, cost-effective and simple point-of-care diagnostic tests for the detection of BV is of utmost importance.
The term "non-specific vaginitis" was the term initially used to distinguish this syndrome from the specific vaginitides caused by Trichomonas vaginalis and yeast (i.e., Candida species). Prior to 1955, the causes of non-specific vaginitis were through to be a wide variety of aerobic bacteria. In 1955, it was reported that Haemophilus vaginalis was the cause of this disease (Gardner and Dukes, Am. J. Obstet. Gynecol., 69:962 (1955)). Subsequently, it was found that the specific organism had no absolute requirement for hemin and, thus, the name was changed to Corynebacterium vaginalis.
Also in 1955, a study was published by Gardner and Dukes which suggested that Gardnerella vaginalis was the causative agent of BV and, thus, the organism thought to be responsible for BV was renamed Gardnerella vaginalis. This theory was, however, discredited by subsequent studies revealing that this microbe, i.e., G. vaginalis, is present in the vaginal secretions of 40-50% of normal women, i.e., BV-negative women, and in those cured of BV (Dunkelberg, et al., Obstet. Gynecol., 20:629 (1962)). As such, the considerable overlap in the levels of G. vaginalis found in BV-positive and BV-negative women has rendered the G. vaginalis cell level inconclusive evidence of the disease state (Amsel, et al., Am. J. Med., 74:14 (1983).)
Since then, it has become apparent that, unlike other common infectious diseases, BV cannot be attributed to one specific etiologic agent, but instead results from the drastic alteration of the vaginal flora. The normally present aerobic Lactobacilli (i.e., the normal flora) become greatly reduced in number and there is a concomitant overgrowth of several anaerobic bacteria and other microorganisms, including G. vaginalis. This alteration in vaginal flora is accompanied by an increase in vaginal pH. In response to these findings, the term BV was introduced to describe this symptomatology, i.e., to describe increased vaginal discharge without signs of clinical inflammation resulting from a complex change in vaginal bacterial flora.
Although BV is the most common form of vaginitis, it is the most benign in symptomatology. The primary signs and symptoms of BV are increased vaginal discharge, genital malodor, increased pH and the presence of clue cells. Normal vaginal discharge is white and floccular, with a high viscosity. With BV, the discharge is non-adherent, clear, thin, white or yellow grey, homogeneous, non-viscous, and watery. Moreover, vaginal discharge from women with BV or trichomoniasis liberates a fishy, amine-odor when the vaginal fluid is mixed with 10% potassium hydroxide (KOH). No such odor is liberated with normal vaginal discharge or, with vaginal discharge from women with vulvovaginal candidiasis. In addition, the vaginal pH of women with BV or trichomoniasis is above 4.5, whereas the normal vaginal pH is less than 4.5. Finally, BV is often associated with the presence of "clue cells," i.e., vaginal epithelial cells to which a large number of bacteria (e.g., G. vaginalis, Mobiluncus species, etc.) are attached rendering the entire cell border obscure.
As such, the clinical "gold standard" method of diagnosing BV involves the examination of the following four vaginal fluid criteria: the presence of clue cells (greater than 20%); vaginal secretions which are white or gray, homogenous and with low viscosity; a vaginal fluid pH greater than 4.5; and fishy or fish-like amine-odor when the vaginal fluid is mixed with 10% KOH. While these tests require relatively inexpensive components to perform, they are not routinely employed in a clinical setting. Such tests are cumbersome, inconvenient, labor intensive, and time-consuming. More importantly, these tests are somewhat subjective and require the health care professional to have considerable expertise with the microscope, a tool not always available in clinics or offices.
In addition to the gold standard criteria, BV is sometimes diagnosed by assessing the shift in vaginal flora by examining Gram stained vaginal smears. This method, however, is difficult to perform and requires special training, thereby making it unsuitable for use in clinic or office settings. Alternatively, a sample of vaginal secretions can be sent to a laboratory for gas-liquid chromatographic analysis for the presence of short chain fatty acids and amines. Unfortunately, however, gas-liquid chromatography is time-consuming and expensive to perform.
In 1988, a report by Thomason, et al. (Obstet. Gynecol., 71(4):607 (1988)) suggested that bacterial enzyme activity, specifically proline iminopeptidase activity, in vaginal fluid may be a suitable marker for BV. Thomason, et al. described a colorimetric assay for proline iminopeptidase activity requiring saline extraction of vaginal fluid from a clinical swab, centrifugation, and a four hour incubation at elevated temperature (37.degree. C.). The enzyme catalyzes the following reaction: ##STR1## The beta-naphthylamine, in turn, is allowed to react spontaneously with a solution of a yellow dye, Fast Garnet GBC, for 5 minutes to produce a red color: ##STR2##
Unfortunately, the proline iminopeptidase assay system described by Thomason, et al., supra, requires four steps to perform. The first step involves the collection of vaginal fluid specimens on standard clinical swabs, and the freezing of the specimens until a sufficient number are available to test concurrently. As the second step, the swabs are thawed, the vaginal fluid is eluted from the swabs with saline, and the extracts are centrifuged to concentrate the insoluble, particulate matter into a pellet. If proline iminopeptidase activity is present in the specimen, it will be present in the particulate matter. As the third step, the pellet is resuspended in Tris buffered saline at pH 7.0 containing L-prolyl-.beta.-naphthylamide and incubated for four hours at elevated temperature (37.degree. C.). During this incubation period, the substrate will hydrolyze to release .beta.-naphthylamine if proline iminopeptidase activity is present in the sample. As the fourth step, a freshly prepared solution of Fast Garnet is added to the suspension, and the mixture incubated for five minutes. If .beta.-naphthylamine has been released by proline iminopeptidase or any enzyme having proline iminopeptidase activity, a red color is formed. In the absence of proline iminopeptidase activity, only the yellow color of the unreacted Fast Garnet chromogen is seen. The resulting assay as a whole is cumbersome, labor intensive, time-consuming and not suitable for use in a clinic or office.
The association of vaginal fluid proline iminopeptidase activity with BV has also been documented by Livengood, et al. (Am. J. Obstet. Gynecol, 163:515 (1990)) using an assay similar to that used by Thomason, et al., supra. Another study by Schoonmaker, et al. (J. Obstet. Gynecol., 165:737 (1991)) utilized a different chromogenic substrate (i.e., L-prolyl-para-nitroanilide) to detect proline iminopeptidase activity in the vaginal fluid from women with BV and normal control women. In this study, the procedure described by Thomason, et al., supra. was performed concurrently on the specimens for purposes of comparison. The results obtained with the second chromogenic substrate produced diagnostic efficiencies (i.e., sensitivity, specificity, positive predictive value and negative predictive value) very similar to those seen in the Thomason study, supra.
The Schoonmaker, et al. procedure requires the following steps: (1) elution of vaginal fluid from clinical swabs and freezing the eluates at -70.degree. C. until the tests were performed; (2) thawing the specimens and concentrating the particulate material by centrifugation; (3) resuspending the pelleted materials and pipetting aliquots into mitrotiter wells; (4) adding the chromogenic substrate to the microtiter wells and incubating the mixture at 37.degree. C. for 4 hours; and (5) determining the presence or absence of a yellow color visually. Thus, as with the Thomason, et al. procedure, the Schoonmaker, et al. procedure is cumbersome, labor intensive, time-consuming and not suitable for use in a clinical or office setting.
In contrast to the foregoing, the ideal BV test for point-of-care use would have the following attributes: (1) room temperature stability to permit convenient storage in patient examining rooms; (2) the ability to use unprocessed or minimally processed vaginal fluid taken directly from the clinical swab; (3) rapid test results, immediately available to guide therapy or monitor therapy; (4) simple, specimen activated format and interpretation without multiple steps or components--ideally, the user would only be required to touch the unprocessed swab to the test system and check for color formation; (5) accuracy equal to that seen with clinical laboratory systems; and (6) built-in, specimen-activated positive and negative control elements to assure proper test performance.
To date, however, no convenient, simple, point-of-care assay has been developed for detecting the presence of enzymatically active proline iminopeptidase, or of any enzyme having proline iminopeptidase activity, in an unprocessed or minimally processed vaginal fluid specimen. Accordingly, the present invention overcomes the problems and disadvantages of the prior art and which has the attributes set forth above for the ideal BV test. Further, the methods and test devices of the present invention are also useful for assaying for the presence of other known hydrolases and hydrolase inhibitors present in unprocessed or minimally processed samples or specimens.